Envelope glycoprotein gp120 contains broadly reactive neutralizing epitopes, which are targeted by human neutralizing antibodies and depend on the native protein conformation. Based on the three dimensional structure, we have identified an insert acceptor site at amino acid 42 of gp120, where foreign protein sequences can be inserted without disrupting the native structure. Our goal was to use this site to enhance the intrinsic vaccine potency of gp120, while retaining important conformational sites. By inserting a strong immunogen, such as the hepatitis B core antigen, at this site, we can combine the immunogenicity of core antigen with the gp120 structure needed to elicit broadly reactive neutralizing antibodies to HIV. Hybrid proteins were expressed in a recombinant vaccinia vector and showed dual antigenicity, normal glycosylation, and high affinity binding of CD4, indicating native folding. By priming with the insert and boosting with the core/env hybrid, we have demonstrated a 20-fold increase in the response to gp120, due to carrier effects. We have now modified the hybrid in order to find one that can self assemble into core antigen-like particles. As observed for other particulate vaccines, these are anticipated to gain up to 1,000-fold greater potency, as compared to monomers. We produced a series of hybrid proteins, linking together each of three different leader sequences with core antigen and two different-sized gp120 sequences. We have found one combination that spontaneously assembles into 25nm particles, as shown by size-exclusion chromatography and electron microscopy. Each particle contains about 180 copies of the core/env hybrid. These multimeric particulate antigens will be tested for immunogenicity in mice and monkeys. If successful, the monkeys will be challenged with titered stocks of SHIV challenge virus. This project was funded by an intramural NIH grant.